Method for exposing peptides and polypeptides on the cell surface of bacteria

ABSTRACT

The inventive method allows peptides or polypeptides to be exposed on the surface of gram-negative host bacteria using specific intimin-based anchor modules. Intimins with shortened carboxy terminals have been found to be particularly suitable anchor modules for passenger domains in the exterior  E.coli  cell membrane. According to said method, host bacteria are transformed using vectors, on which are located a fused nucleic acid sequence consisting of a sequence segment which codes for an intimin with a shortened carboxy terminal and a nucleic acid sequence segment which codes for the passenger peptide that is to be exposed. The invention permits a particularly large number of passenger domains to be exposed on the cell surface of the bacteria, without adversely affecting the viability of the bacteria.

[0001] The present invention concerns a method for exposing peptides,including polypeptides and proteins, depending on the length of thesequence exposed, on the surface of Gram-negative bacterial cells,especially Escherichia coli cells, an accompanying process for producinga variant population of surface-exposed peptides or polypeptides and foridentifying bacteria which carry peptides or polypeptides with aparticular desired property, and vectors and host bacteria which can beused in the process.

[0002] In general, the method allows expression of recombinant proteins,which can be receptors or ligands, on the bacterial surface andselection based on affinity of binding to a binding partner. The methodallows expression of peptide and polypeptide libraries on the surface ofbacterial cells, by means of which peptide molecules with high affinityto a ligand can be identified. The method allows exposure of aparticularly large number of passenger proteins or peptides on thesurface of a bacterial cell without adversely affecting its cellularviability. This method further allows setting the number of moleculespresented on the surface of a cell as desired.

[0003] In particular, the present invention also concerns isolation ofmonospecific antibodies from polyclonal antibody mixtures by binding topeptide epitopes anchored to the cell surface.

BACKGROUND

[0004] Expression of proteins and protein domains on the surface ofself-replicating carriers such as bacteriophages, bacteria, yeasts,etc., is currently under intense investigation. The primary objective isto achieve coupling of the functional expression of a property of theprotein exposed on the surface of the carrier (phenotype) with thefundamental genetic coding (genotype). Examples of successfulphenotype/genotype coupling appear, among others, in the use of yeastsas carriers of surface-exposed proteins. For instance, a molecularlibrary of variant yeast cells is generated, each one of which hasdifferent immunoglobulin fragments exposed on its cell surface. Thosecells which exhibit increased affinity to a specific ligand molecule canbe isolated from that library of antibody variations (Boder and Wittrup(1997), Nat. Biotechnol. 15:1553).

[0005] Bacteria have broad applications for cell surface exposure. BothGram-positive and Gram-negative types are used. For instance, proteinscan be exposed on Staphylococcus xylosus and Staphylococcus carnosus byStaphylococcus aureus protein A. Enzymes can be anchored to the surfaceof S. carnosus by fusion to Staphyloccus aureus fibronectin bindingprotein B (FnBPB) (Strauss and Götz (1996), Mol. Microbiol. 21:491-500).However, Gram-positive bacteria are less suitable for exposure of largepeptide libraries because it is difficult to introduce the correspondinggene variants into the cells by transformation and to generate asufficiently large number of independent clones which differ withrespect to the coding nucleotide sequence of the surface-exposed proteinvariants.

[0006] Gram-negative bacteria, on the other hand, are very well suitedto generation of molecular libraries and to the accompanying exposure ofaltered proteins because of their high transformation yield (>10° permicrogram of plasmid DNA for E. coli), and so are preferred hostorganisms.

[0007] Various systems have been described for exposing recombinantproteins on the cell surface of Gram-negative bacteria (Georgiou et al.(1997), Nature Biotechnol. 15:29-34). In general, surface exposure isattained by fusing gene segments of a bacterial surface protein with thegene for the protein to be exposed. The proteins usually used ascarriers are those which are secreted and/or localized in the externalmembrane of Gram-negative bacteria and therefore contain the signalsneeded for translocation through the cytoplasmic membrane, passage intothe bacterial periplasm, and integration into the external membrane oranchoring on the surface of the external membrane. The carrier proteinsthat have been used most are those which are themselves integralcomponents of the external membrane of E. coli. Those include, amongothers, PhoE (Agterbert et al. (1987), Gene 59:145-150) or OmpA(Francisco et al. (1992), Proc. Natl. Acad. Sci. USA 89:2713-2717); butthere are disadvantages to their use. For instance, protein sequencescan be inserted only into surface-exposed loops of these proteins. Thatresults in conformationally fixed amino and carboxy terminations, anddrastically limits the length of the peptide sequence to be inserted.Use of the peptidoglycan-associated lipoprotein (PAL) as a carrierprotein does indeed result in transport to the external membrane, but itis impossible to expose active and correctly folded protein sequences onthe surface of E. coli (Fuchs et al. (1991), Bio. Technology 9,1369-1372). It has been possible to expose large proteins on the surfaceby (a) use of fusion of a fragment of the Escherichia coli Lpp and ofthe OmpA protein as the carrier protein portion, to the carboxy end ofwhich the passenger protein sequence is attached (Francisco et al.(1992), Proc. Natl. Acad. Sci. 89:2713-2717); (b) use of the IgAprotease (domain (IgAβ) and other bacterial autotransporters (Maurer etal. (1997) J. Bacteriol. 179: 794-804), and (c) by use of the icenucleation protein of Pseudomonas syringae (InaZ) (Jung et al. (1998),Nature Biotechnol. 16:576-580 as the carrier protein portion.

[0008] It is clear from the examples above that proteins can be exposedon the bacterial cell surface by joining a passenger domain to a carrierprotein by fusion of the corresponding coding DNA sequence with thecoding sequence of a selected protein of the external membrane ormembrane protein fragment. In this case the membrane protein or membraneprotein fragment provides the force needed for the membrane localizationand anchoring. Here the carrier protein of the external membrane should(a) have a secretion signal that assures passage through the cytoplasmicmembrane; (b) exhibit a localization signal for embedment into theexternal membrane; (c) appear on the cell surface in the highestpossible number of copies; and (d) not have a negative effect on thestructural and functional integrity and, in particular, the vitality ofthe host cell.

[0009] Substantial problems have been found, though, with the processesdescribed at the state of the art for production of heterologouspassenger proteins using proteins of the external membrane, particularlywith respect to requirements (c) and (d). A high expression ratio and ahigh net accumulation in the external membrane are always accompanied byhigh mortality of the bacterial cells which expose them. For instance,strong over-expression of fusion proteins with Lpp-OmpA as the membraneanchor is lethal (Daugherty et al. (1999), Protein Eng. 12:613-21). Highcell mortality was likewise described for use of the autotranporter ofthe IgA protease (IgAβ) (Wentzel et al. (1999), J. Biol. Chem. 274:21037-21043). Jung et al. Introduced the ice nucleation protein ofPseudomonas syringae, which is a glycosyl-phosphatidylinositol anchoredprotein of the external membrane, into E. coli as a carrier protein forcell surface exposure of passenger proteins (Jung et al. (1998), NatureBiotechnol. 16: 576-580). This carrier protein does allow stableexposure of passengers on the surface of the external membrane; but thefusion proteins aggregate in clusters on the bacterial surface. Thatcharacteristic is undesirable for the purpose of selecting peptides andpolypeptides with high affinity to a specific binding partner.

[0010] Aside from the proteins integral to the external membrane, othersurface structures present on the cell surface, such as flagellae, pili,fimbriae, etc., have been used as carriers for exposure of passengerdomains. Various peptides of Hepatitis B virus were stably expressed andexposed on the bacterial surface by use of flagellin, a subunit of theflagellum, as the carrier (Newton et al. (1989), Science 244: 70-72).However, as for use of fimbrin as a structural carrier protein, exposureof passenger domains remains limited to small peptides (Hedegaard et al.(1989), Gene 85: 115-124).

[0011] Technical Problems, and Their Solution by the Present Invention

[0012] The present invention is, therefore, based on the technicalproblem of providing carrier proteins, which do not result in thedisadvantages stated above, especially with use of Escherichia coli.

[0013] An optimal presentation procedure must meet the followingrequirements:

[0014] 1. The peptide/protein to be exposed should preferably beanchored on the surface of a bacterial cell in the highest possiblenumber of copies.

[0015] 2. The peptide/protein exposure should not impair viability.

[0016] 3. The number of peptide/protein molecules exposed on the surfaceper cell should be controllable within wide limits.

[0017] No method of bacterial surface exposure which meets theserequirements in all points has yet been described.

SUMMARY OF THE INVENTION

[0018] To achieve the objective stated above, a process is provided forexposure of peptides and/or polypeptides on the surface of host bacteriain which one (a) produces a Gram-negative host bacterium which istransformed with a vector to which is localized a fused nucleic acidsequence that (i) has a sequence segment that codes for an Intiminshortened by at least the C3 domain at the carboxy terminus region asthe anchoring domain and (ii) has a nucleic acid segment coding for thepassenger peptide and/or passenger polypeptide to be exposed, and (b)cultivates the host bacterium under conditions in which the codednucleic acid sequence is expressed and the peptide or polypeptide codedby the nucleic acid sequence (ii) is exposed on the surface of the hostbacterium, such that the nucleic acid sequence (ii) is heterologous withrespect to the nucleic acid sequence segment coding for the Intiminmembrane anchoring domain.

[0019] The shortened Intimin can be shorted by at least one more of thedomains D0, D1 and/or D2 in the carboxyterminal region of 280 aminoacids (aside from the shortening by the D3 domain).

[0020] The method of the invention allows exposure of peptides orpolypeptides on the surface of Gram-negative host bacteria using certainIntimin-based anchoring modules. It has been found that Intiminsshortened at the carboxy terminus are particularly well suited asanchoring units in the external E. coli cell membrane for passengerdomains.

[0021] The invention is based on providing a gene construct in which aspecial carrier protein (a fragment of the “Intimin”, see below) is usedas the exposure anchor. To expose a specified peptide/protein, thecoding gene is fused to the coding sequence of the Intimin gene fragmentin the continuous reading frame. Surprisingly, it is found that when anIntimin fragment is used as the membrane anchor a very large number ofmolecules can be exposed in the bacterial membrane without impairing theviability of the host bacteria.

[0022] (b) [sic/tr. note #1] We have succeeded in establishing thenumber of molecules per cell as we wish by combined regulation of theexpression of the Intimin gene at the transcription and translationlevel. Processes previously described for regulating gene expression forthe purpose of establishing surface-exposed molecules do not do that.

[0023] Intimin as the Exposure Anchor

[0024] The further development of the invention provides that theIntimin anchoring domain in the external bacterial membrane be derivedfrom the genus of the Enterobacteriaceae and used in a host bacterium ofa genus of the Enterobacteriaceae [sic/tr. note #2]. It is furtherpreferable for the anchoring domain to be a fragment of the Intimin ofenterohemorrhagic E. coli or a variant of it. At present acarboxy-terminal shortened variant of the BacA Intimin fromenterohemorrhagic Escherichia coli O157:H7 comprising amino acids 1 to659 or, alternatively, amino acids 1 to 753, is particularly preferred.

[0025] Many strains of pathogenic bacteria have surface structures suchas pili, glycoproteins and other proteins, such as homopolymeric andheteropolymeric carbohydrate glycocalices, by means of which thebacteria adhere to surfaces of eucaryotic cells. Proteins of thebacterial cell surface called Intimins play an important part in thefirm adhesion of enteropathogenic (EPEC) or enterohemorrhagic E. coli(EHEC) bacteria to the surfaces of eucaryotic host cells. TheIntimin-mediated adhesion allows the bacteria to multiply on thesurfaces of the colonized host cells. Intimin is a member of the familyof bacterial adhesion molecules which have sequence homologies with eachother in the area of the amino-terminal region (McGraw et al. (1999),Mol. Biol. Evol. 16:12-22).

[0026] Intimin is the product of the Eae gene. It has 939 amino acidgroups. Its cell-binding activity is localized in the 280 C-terminalamino acid groups. Intimin is anchored in the external membrane with itsamino-terminal domain, which exposes the 280 carboxy-terminal aminoacids. These 280 groups code for three domains, two immunoglobulin-likedomains and one lectin-like domain (Kelly et al. (1999), Nat. Struct.Biol. 6:313-318) code for these 280 groups, which are responsible forbinding to eucaryotic cells. The structures of domains D1, D2 and D3 areknown, but not the structure of the amino-terminal domain (Kelly et al.(1999) Nat. Struct. Biol. 6:313-318). FIG. 1 shows the schematicstructure of Intimin and its anchoring in the external membrane.Corresponding domains of other Intimins can be derived from sequencecomparisons.

[0027] Except for the 200 amino-terminal amino acid groups, Intiminsexhibit sequence homologies with invasins from Yersinia and otherbacteria which make it possible for the bacterium to enter cultivatedmammalian cells through binding with integrins (Leong et al. (1990),EMBO J. 9:1979-1989). Integrins and invasins do, to be sure, havesimilar sequences, but they are assigned to different protein familieswith respect to their functions (Batchelor et al. (2000), EMBO J.19:2452-2464).

[0028] Surprisingly, surface presentation of peptides or polypeptidesdistinctly improved over the state of the art was attained by use of afragment of Escherichia coli Intimin as the transporter domain forbacterial surface localization of passenger proteins and passengerpeptides, particularly also of short synthetic peptides having lengthsof preferably 6 to 20 amino acids, by disulfide-bridged peptides andpolypeptides, especially oligopeptides on the structural basis of thecystine node folding motif (Pallaghy et al. (1994), Protein Sci. 3:1833-1839), or by bacterial (e. g.: (-lactamase inhibitor protein) andeucaryotic polypeptides (e. g., interleukin 4).

[0029] Identification of Eae Intimin as the Carrier Protein for SurfaceExposure of Passenger Proteins

[0030] The Escherichia coli Intimin that is used preferably is localizedin the external membrane of Escherichia coli. It naturally exposes atleast one protein domain on the outer side of the external membrane. TheEscherichia coli Intimin is anchored in the external membrane, andcarries at its carboxy-terminal end four domains which are necessary forinteraction with a receptor protein on the surface of epithelial cells.

[0031] Beginning with the working hypothesis that substitution of atleast one of the Intimin domains exposed on the cell surface by aheterologous passenger domain would result in that domain being exposedon the surface, a gene fusion was produced from a nucleic acid segmentcoding for a passenger protein and one coding for an Intimin fragment.Example 1 presents the gene fusion from the Intimin fragment and thepassenger domain, the vector construction, and the expression in hostbacteria, compared with other surface presentation methods.

[0032] In general, the Intimin gene or gene fragment selected for genefusion is amplified by the polymerase chain reaction and cloned in avector suitable for expression in the intended host bacterium. A genefor a passenger peptide to be exposed is introduced into the same genedownstream from the selected Intimin fragment in the same reading frame.The vector also contains an exogenously inducible promoter operativelylinked with the Intimin gene and with the passenger peptide fused withit. Other functional sequences, such as marker genes, can also bepresent.

[0033] In further development of the invention, the process is arrangedso that the expression of the fusion gene from the nucleic acid sequencesegment coding for the shortened Intimin and for the passenger proteincan be regulated by (i) replacing a codon coding for a glutamine in theshortened nucleic acid sequence segment coding for the shortened Intiminby an amber stop codon (TAG), and (ii) using an Escherichia coli hoststrain in which a translation of the mRNA of the fusion gene isaccomplished by providing a controllable quantity of suppressor tRNA,which allows over-reading of the stop codon on translation. Thisprocedure allows effective regulation of the expression of the segmentexposed within the host cell, and is useful for practically all knownexposure processes.

[0034] In the further developed process a CAG codon within the Intiminfragment is replaced by a TAG stop codon. That is accomplished, forinstance, by cloning a PCR fragment in which the CAG codon #35 in theIntimin is replaced by a TAG stop codon. By use of an amber suppressorstrain, this stop codon is over-read and a glutamine group isincorporated at this position. As the efficiency of amber suppression islow, fewer molecules are synthesized than in the absence of the stopcodon. The result is that the number of surface-exposed moleculesremains within an extent that is tolerable for the cell (in thisconnection, see Christmann et al. (1999), Protein Eng. 12: 797-806). Anyexpression vector particularly usable for expression in E. coli can beused. The vector pASK75 (see below), among others, is a suitablestarting vector.

[0035] Regulation of the Gene Expression

[0036] Expression of a gene is usually regulated by the coding sequenceof a gene being brought under control of a promoter such that the numberof transcriptions per unit time can be regulated by the concentration ofan inducer molecule added exogenously. For instance, the lac promoter,the ara promoter, or the tetA promoter can be considered for thatpurpose (Lutz & Bujard (1997), Nucleic Acids. Res. 25:1203). It has notpreviously been possible satisfactorily to regulate gene expression withthe goal of establishing a desired number of surface-exposed moleculesper bacterial cell by controlled induction of the transcription of thegene for surface-exposed fusion proteins. Variation of the concentrationof an added inducer has not previously resulted in accumulation offusion proteins on the surface of the bacterial cell depending on theconcentration of the inducer (Daugherty et al. (1999), Protein Eng. 12:613-621). A slight improvement was gained by adjusting the netaccumulation by means of the induction period. That means that thetranscription inducer was added to a bacterial culture, and the cellswere incubated with the inducer for different times. The longer theinduction time, the higher the number of surface-exposed cells per cell[sic/tr. note #5]. This process is not suitable for highly parallelbiotechnological applications, as it requires sample collection atdifferent growth times. Also, the cells are in different physiologicalstates, depending on the growth time and the absorbance [sic/tr. note#7] attained.

[0037] The process which we have developed eliminates this disadvantage.The gene expression is controlled on two levels, the level oftranscription and the level of translation.

[0038] In one process according to the invention, fusion proteins areproduced by replacing a codon coding for glutamine (CAG) in the nucleicacid sequence coding for the Intimin or Intimin fragment by a amber stopcodon (TAG). This codon is preferably the first glutamine codon of theamino acid sequence of Intimin or the Intimin fragment. Likewise, adifferent codon within the first 100 amino acids of the Intimin orIntimin fragment can be replaced by TAG. Now a modified E. coli strainis offered in trans a modified glutaminyl-tRNA, which carries thegenetic marker supE. This supE tRNA is able to pair in the translationwith a TAG codon, and to cause suppression of the translation stop.

[0039] An E. coli host bacterium which contains the nucleic acidsequence segment coding for the supE gene in operative linkage with acontrollable promoter is according to the invention. The PI lacpromoter, with which the intensity of expression and thus the rate ofsynthesis of supE transfer RNA can be controlled by the amount of theinducer IPTG added to the growth medium for the host bacteria, ispreferred. In one typical example, the nucleic acid sequence segment,which codes for a supE tRNA gene in operative linkage with acontrollable promoter is localized in a vector compatible with theexpression vector. Transcription of the gene for the Intimin passengerdomain fusion protein is switched on by addition of an inducer(anhydrotetracycline in this case). Different, and freely adjustable,quantities of suppressor tRNA for synthesis of the Intimin fusionprotein are made available in the cell by varying the amount of IPTGinducer. Finally, the amount of supE tRNA determines the average numberof passenger domains exposed on the surface of a bacterial cell. FIG. 5shows schematically the newly developed expression process.

EMBODIMENTS

[0040] The process according to the invention produces a host bacteriumtransformed with one or more compatible vectors. Such a vector containsa fused nucleic acid sequence in operative linkage with a promoter andoptionally other sequences needed for the expression. This fused nucleicacid sequence includes (a) a nucleic acid sequence segment coding for anIntimin fragment which makes possible exposure of the peptide orpolypeptide coded by segment (b) on the outside of the external membraneof the host bacterium and (b) a nucleic acid sequence segment coding forthe protein and/or peptide to be exposed.

[0041] In one preferred embodiment, then, the present invention concernsan Intimin, a fragment of Intimin, or a carrier protein homologous withIntimin, which exerts a transporter function and allows surface exposureof recombinant proteins in the host bacteria in high numbers of copies.This involves the amino-terminal fragment of the Eae Intimin fromenterohemorrhagic Escherichia coli Serotype O157:H7 (Louie et al.(1993), 61:4085-4092) which extends from amino acid 1 to 659. Along withthis specific sequence, the invention also covers use of variants, whichcan, for example, be produced by alteration or deletion in the aminoacid sequence in the sequence segments not essential for translocationthrough the cytoplasmic membrane and localization in the externalmembrane.

[0042] Another typical example involves gamma Intimin from E. coli (GeneBank Accession Number AF081182), Intimin from E. coli O111 :H— (GeneBank Accession Number AAC69247) or Intimin from other Escherichia coliserotypes or the Intimin from Citrobacter freundii (Gene Bank AccessionNumber AAA23097) as the transporter protein used. The DNA sequences andthe amino acid sequences derived from them for the Intimins listed abovecan be found as NCBI citations (National Center for BiotechnologyInformation, USA) at the locations listed below.

[0043] Other Intimin domains can be derived from protein sequences indatabases, from protein sequences based on DNA sequences available indatabases, or from protein sequences determined by sequence analysisdirectly or indirectly from the DNA sequence. The corresponding codingregions (genes) can be used to produce vectors or fusion protein genes,which make possible effective surface expression of passenger proteinsin Gram-negative bacteria, especially Escherichia coli.

[0044] In the invention, surface presentation or exposure means that thefusion proteins or passenger domains are localized on the side of theexternal bacterial membrane toward the medium. Surface-exposed passengerproteins in intact Gram-negative bacteria are freely accessible forbinding partners.

[0045] In one preferred embodiment, the present invention thus enablessurface exposure of peptides or, in a further embodiment, the surfaceexposure of peptide libraries in Gram-negative bacteria, especially inE. coli, and their use to determine affinity to an antibody or anotherreceptor.

[0046] In another preferred embodiment, the present invention makespossible mapping of epitopes and isolation of monospecific antibodiesfrom an antibody mixture. Epitope mapping means that the peptide withthe highest affinity to an antibody or another receptor, exposed on thesurface of the producing strain, is identified. That makes clear acritical advantage of the present invention for expression of peptidelibraries, compared with the phage systems used for such applications(Makowski (1993), Gene 128: 5-11; Kuwabara et al. (1997), Nat.Biotechnol. 15: 74-78). In the bacterial system according to theinvention, selection of the clonal producers occurs simultaneously withidentification of a peptide having the desired binding property. Theycan be multiplied immediately. In one typical example, clonaldescendants of the producers were used to purify or isolate an antibodyor another receptor from a mixture of molecules. That occurs throughbinding of the receptor molecules with high affinity to the peptidemolecules exposed on the bacterial surface, followed by separation ofthe unbound molecules by centrifugation and/or filtration, andseparation of the monospecific antibodies or receptor molecules from thesurface-exposed peptides.

[0047] Multiplication of the strain expressing the desiredsurface-exposed peptide or protein accomplishes amplification of thecorresponding coding gene. Sequence analysis of that gene allowsunambiguous and simple identification and characterization of thepeptide or protein. Thus a peptide library prepared in that mannercontains fusion proteins, made up of an Intimin or an Intimin fragmentand a peptide or protein produced and surface-exposed in a Gram-negativebacterium, preferably E. coli. In one typical example, cloning ofsynthetic oligonucleotides degenerated at selected positions behind thecoding sequence of Intimin or Intimin fragments achieves the highvariance of the different expressed proteins.

[0048] In one particularly preferred embodiment, the process accordingto the invention makes possible surface expression and variation of apeptide or polypeptide having an affinity to a binding partner, aligand, a receptor, an antigen, a protein with enzymatic activity, anantibody, or an antigen-binding domain of an antibody.

[0049] The process according to the invention for producing a variantpopulation of surface-exposed peptides and for identification ofbacteria, which carry the peptides or polypeptides with a desiredproperty, is organized in the following steps:

[0050] 1) Production of at least one fusion gene by cloning the codingsequence of a desired passenger in the continuous reading framedownstream from an Intimin gene or an Intimin gene fragment in at leastone vector.

[0051] 2) Variation of the passenger by cloning passengers from a genemixture, or through site-directed mutagenesis, e.g., by the polymerasechain reaction (PCR) using oligonucleotides with deliberately replacedbases, by random mutagenesis using oligonucleotide mixtures withrandomly produced base sequences in selected sequence segments in thePCR, by error-prone PCR, by randomly controlled chemical mutagenesis, orby use of high-energy radiation.

[0052] 3) Incorporation of the vector or vectors in host bacteria.

[0053] 4) Expression of the fusion gene in the host bacteria, which thenexpress the fusion protein stably on their surfaces.

[0054] 5) Cultivation of the bacteria in liquid culture or on agarplates for clonal expansion.

[0055] 6) Optional selection of the bacteria which carry the passengerwith the desired properties, and

[0056] 7) Optional characterization of the selected passenger throughsequencing of the nucleic acid sequence segment coding the passengerpeptide or polypeptide.

[0057] 8) Optional isolation and purification of a binding partner forthe passenger with the optimal properties.

[0058] This process can be carried out repetitively.

[0059] In one preferred embodiment of this process, the bacteria havinga stable exposed fusion protein with the desired properties are isolatedby binding to an immobilized and/or labeled binding partner, e. g., amatrix-fixed binding partner, a magnetic-particle-labeled bindingpartner, or a chromogenically or fluorogenically labeled bindingpartner.

[0060] Bacterial Surface Exposure of Protein Fragments, Epitope Mappingand Isolation of Monospecific Antibodies.

[0061] Every protein carries many antigenic determinants on its surface.As a result, the immune response to such a molecule is alwaysstimulation of many B-lymphocytes and production of just as manyantibody species. Knowledge of the amino acid sequence of such epitopesis of great importance for immunological research.

[0062] It would be advantageous for many applications if one couldobtain large amounts of monospecific antibodies from mapped serumimmediately after mapping of epitopes. Monospecific antibodies areequally monoclonal in their properties, and are also sold commercially.To isolate such antibodies, the peptides, which comprise the epitope,must be coupled to a matrix. Then the serum is passed over that matrix,and the antibodies, which bind specifically to the desired antigen, areeluted. In this example of the application of the Intimin-basedbacterial surface exposure, epitope-presenting E. coli cells were usedas such a matrix.

[0063]FIG. 9 shows a survey of the procedure in epitope mapping usingIntimin-mediated cell surface exposure. Example 2 presents the use ofthis process.

[0064] Isolation of Peptides with Affinity to a Specific Target ProteinThrough Intimin-Based Surface Exposure of Combinatory Peptide Libraries

[0065] To check whether Intimin-based bacterial surface exposure issuitable for isolating from a molecular collection of surface-exposedpeptide variants those which have affinity to a specified targetprotein, a library of variants of the cystine node protein EETI-II,comprising 28 amino acids, was generated. EETI-II is an inhibitor oftrypsin proteases which occurs in the vegetable marrow Ecballiumelaterium. This peptide is stabilized by three intramolecular disulfidebridged which spread out a group of surface loops (described in Wentzelet al. (1999), J. Biol. Chem. 274: 21037-21043). A library of EETI-IIvariants was generated, in which the residues of two loop regionsexposed to the solvent are randomized. Example 3 presents theexperimental procedure for this example embodiment.

[0066] The invention is explained in more detail in the following usingsome experimental examples which will make it easier to understand theinvention, but which are not intended to limit the invention to theseexamples. Those skilled in the art will recognize which alternatives arepossible within the outlines of the invention on the basis of theseexamples.

BRIEF DESCRIPTION OF THE FIGURES

[0067] The accompanying figures will serve for further explanation ofthe invention. Individually, they show:

[0068]FIG. 1: Schematic representation of the structure of an Intimin.OM: external membrane; D0 to D3: extracellular domains 1 to 4. TheIntimin fragment preferred here, which serves as the carrier forpassenger domains, lacks domains D2 and D3.

[0069]FIG. 2: Schematic representation of the expression vectorpASK-INT-EETI-CKSend. Intimin: coding sequence for the Intimin genefragment; etag: coding sequence for an epitope sequence (Etag); ead:coding sequence for the EETI-II microprotein; cat: gene forchloramphenicol acetyltransferase; tetR: gene for Tet repressor; bla:gene for β-lactamase.

[0070]FIG. 3: Flow-cytometric analysis of the bacterial surface exposureof an Intimin-EETI-II microprotein fusion protein.

[0071]FIG. 4: Survival proportions of E. coli cells which expose amicroprotein on the bacterial cell surface by means of the Intiminmembrane anchor.

[0072]FIG. 5: Schematic representation of control of gene expressionthrough supE-mediated translation control.

[0073]FIG. 6a: Nucleotide sequence and genetic organization of thesequence segment of the E. coli genome fromBMH71-18 amplified by the PCRprimers SupE2-Eco-up and SupE2-Mlu-lo. The tRNA coded genes areemphasized in bold type.

[0074]FIG. 6b: Schematic representation of the vector pZA22-MCS1 (Lutz &Bujard (1997), Nucleic Acids Res. 25:1203) and of the vector pZA22-supEresulting from incorporation of the supE gene. PL-lac-O1: hybridoperator/promoter region from the PL operator and the lac promoter; RBS:ribosomal binding site; MCS-1: polylinker sequence; P15A: replicationorigin; KanR: Kanamycin resistance gene.

[0075]FIG. 7: Schematic representation of the vector pREP4-supE.Plac-supE: hybrid operator/promoter region of the PL operator and thelac promoter (Lutz & Bujard (1997) Nucleic Acids Res. 25:1203) followedby a sequence segment containing the supE gene. P15A: replicationorigin; KanR: kanamycin resistance gene; lacI: gene for the lacrepressor.

[0076]FIG. 8: Regulation of the synthesis and surface exposure ofIntimin-anchored passenger domains by controlled provision of supE-tRNA.

[0077]FIG. 9: Procedure for mapping of linear peptide epitopes.

[0078]FIG. 10: Representation of the two linker molecules resulting fromhybridization of 4 oligonucleotides, which generate the corresponding,vector cleavage sites (AvaI/BamHI).

[0079]FIG. 11: Position of the epitope identified in the proteinsequence of PMS1.

[0080]FIG. 12: A) PMS1_KL6 labeled with purified antibodies againstclone 7 (1) and with purified antibodies against its own epitope (2);

[0081] B) PMS1_KL7 labeled with purified antibodies against clone 6 andwith purified antibodies against its own epitope (2).

[0082]FIG. 13: Amino acid sequence of the library of EETI-II variants.Amino acids in the single letter code. The two loop regions in whichamino acid positions (X) are randomized are indicated in italics

[0083]FIG. 14: Enrichment of the microproteins that bind theanti-β-lactamase antibodies. All of the cells were labeled withanti-β-lactamase as described in the text.

[0084] A) Unselected library labeled with anti-β-lactamase serum,anti-rabbit antibodies (biotin conjugate) and streptavidin-Rphycoerythrin. 1.6% of the cells appear in the window used (fluorescencechannels 300-1024).

[0085] B) Second round of sorting (labeling as in A);

[0086] c) Third round (labeling as in A).

[0087]FIG. 15: Amino acid sequence of the 6 EETI variants, whichinteract with the anti-β-lactamase antibodies; randomized amino acidsare underlaid with gray. Underlines indicate a mutation from the wildtype gene in the DNA sequence. A consensus sequence for binding to theanti-underlaid with gray. Underlines indicate a mutation from the wildtype gene in the DNA sequence. A consensus sequence for binding to theanti-β-lactamase antibody is shown for the rear loop region.

EXAMPLES Example 1 Gene Fusion of a Nucleic Acid Sequence Segment Codingfor a Passenger Protein and One Coding for an Intimin Fragment VectorConstruction, and comparative Example

[0088] The cystine node polypeptide EETI-II, a trypsin proteaseinhibitor comprising 28 amino acids, was selected as the passenger forthe first example (as previously used by: Wentzel et al. (1999), J.Biol. Chem. 274: 21037-21043; Christmann et al. (1999), Protein Eng. 12:797-806).

[0089] For that purpose, the Intimin gene eae (Gene Bank AccessionZ11541) from EHEC O157:H7 Strain 933 bacteria was amplified with thepolymerase chain reaction using the oligonucleotides Intiminup andIntiminlo. EHEC O157:H7 bacteria inactivated by boiling were used as thetemplate. The gene was amplified with the following amplificationconditions: 30 sec 94° C., 30 sec 53° C. and 2 min 72° C., 30 cycles.

[0090] The oligonucleotide Intiminup hybridizes the eae gene fromnucleotide 69 to 88. This sequence segment contains the ATG start codonand the 17 nucleotides downstream from it. This oligonucleotide contains5′ additional sequences which [represent/tr. note #6] cloning of the PCRproduct on the 5′ side into the Xbal cleavage site of the vectorpASK21TE-EETI-CKSend (Christmann et al. (1999), Protein Eng. 12:797-806). The oligonucleotide Intilo1 hybridizes the eae gene fromnucleotide 2028 to 2045 and contains at the 5′end the coding sequencefor a MluI cleavage site which allows cloning at the 3′ end in the EcoRIcleavage site of vector pAsk21-EETI. Thus the PCR product contains thecoding nucleic acid sequence for codons 1 to 659 of the Intimin. Then atripartite fusion gene in operative linkage with the tetApromoter/operator is localized in the resulting vector pASK-Inti-EETI.It consists of (a) the coding nucleic acid sequence for Intimin fromCodon 1 to 659; (b) the coding nucleic acid sequence for an epitope fromthe human Gla protein comprising 17 amino acids (Etag; Kiefer et al.(1990), Nucleic Acids res. 18: 1909) and (c) the coding nucleic acidsequence for the cystine node polypeptide EETI-CKSend (Christmann et al.(1999), Protein Eng. 12: 797-806; FIG. 2).

[0091] By cloning a PCR fragment, the CAG codon number 35 in Intimin wasreplaced by a TAG stop codon.

[0092] A second plasmid (pASK21TE-EETI-CKSend) in which the Intiminsequence is replaced by a shortened OmpA fragment (Christmann et al.(1999), Protein Eng. 12:797-806) was used to be able to compare theexpression level with previous processes for surface exposure.

[0093] The strain BMH71-18 (Christmann et al. (1999), Protein Eng. 12:797-806) was used as the bacterial strain for the gene expression.

[0094] BMH71-18: [F¹laol^(q) lacZΔM15, proA+B+; Δ(lao-proAB), supE, thi]

[0095] The transformed cells were grown at 37° C. to an absorbance of0.2 to 0.4. Expression of the fusion gene was induced by addition of 0.2ug/ml anhydrotetracycline. The surface exposure was demonstrated bybinding of a monoclonal anti-Etag antibody to the surface of the cellsby means of indirect fluorescence labeling. That was done bycentrifuging the cells off after one hour of incubation and incubatingthem successively with monoclonal anti-Etag antibody, biotinylatedanti-mouse antibody and streptavidin, phycoerythrin conjugate exactly asdescribed by Christinann et al., (1999), Protein Eng. 12: 797-806. Thelabeled cells were measured in a flow cytometer (FIG. 3). The FACSdiagram shows the fluorescence of cells which produce no fusion protein(negative control, A), those which produce the Intimin-Etag-EETI fusionprotein (B) and, for comparison, (C) cells transformed with a plasmid inwhich the Intimin membrane anchor has been replaced by an Ipp-OmpAmembrane anchor corresponding to the previous Sder technique.

[0096] As can be seen from this figure, the cells in whichIntimin-mediated exposure of the fusion partner occurs exhibit aboutten-fold higher fluorescence than the cells with the usual membraneanchor. This finding shows, surprisingly, that there are about ten timesmore molecules of the passenger protein available on the bacterialsurface when the Intimin membrane anchor is used. Furthermore, theviability of the cells, which is strongly reduced in the previouslydescribed processes for over-expression of membrane-anchored passengerproteins, is not notably impaired by the overproduction of the fusionprotein from the Intimin fragment and the passenger domain. The survivalproportions of the cells before and 1, 2 and 4 hours after induction ofthe expression of the fusion protein is shown below (FIG. 4).

[0097] Combined use of supE tRNA in Host Organisms and the Amber StopCod n in the Intimin Fragment

[0098] The CAG codon number 35 in Intimin was replaced with a TAG stopcodon by cloning a PCR fragment. When an amber suppressor strain isused, this stop codon is over-read and a glutamine is incorporated atthat position. As the efficiency of the amber suppression is low, fewermolecules are synthesized than in the absence of the stop codon. Thatmakes sure that the number of surface-exposed molecules remains withinan extent that is tolerable for the cell (in this respect, seeChristmann et al. (1999), Protein Eng. 12: 797-806). FIG. 2 shows thenucleotide sequence of the 5′-terminal and the ‘3’ terminal part of theIntimin gene in the expression vector pASK-INT-EETI-CKSend, with theamber stop codon #35 emphasized in bold face.

[0099] The amber suppressor strain BMH71-18 was used as the PCR templateto obtain the supE.

[0100] BMH71-18: [F¹ laol^(q) lacZΔM15, proA+B+; Δ(lao-proAB), supE,thi]

[0101] The gene was amplified from the genome under standard conditionsusing the primers SupE2-Eco-up and SupE2-Mlu-lo. FIG. 6a shows thesequence of the genome segment and of the primer.

[0102] The two PCR primers SupE2-Eco-up and SupE2-Mlu-lo were used foramplification. The supE gene lies in a cluster of tRNA genes, which aretranscribed polycistronically and then processed and liberated byspecific RNAses. The supE gene and at least one additional tRNA geneupstream and downstream were amplified with these oligonucleotides. Theprimers also introduce an EcoRI- and a MluI cleavage site. The resultingDNA segment was hydrolyzed with EcoRI and MluI and placed in the vectorPZA22-MCS1 split with EcoRI and MluI (Lutz & Bujard (1997), NucleicAcids Res. 25:1203), as shown in FIG. 6b.

[0103] The sequence segment coding for the supE gene appears in thisvector, again under the control of the P<lacO-1 promoter/operator MCS1(Lutz & Bujard, Nucleic Acids Res. 1997, 25:1203) inducible with IPTG.The promoter/operator and the supE gene sequence segment downstream fromit were removed by splitting the resulting plasmid with XhoI and XabI.The ends were filled out with T4 polymerase in the presence of dATP,dGTP, dCTP and dTTP. The resulting fragment was inserted into the vectorpRep4, split with SmaI (Qiagen). FIG. 7 shows a schematic map of theresulting vector.

[0104] Transcription of the supE gene from pREP4-supE can be induced byIPTG induction, thus inducing formation of supE-tRNA, which can becontrolled by varying the quantity of the inducer. The plasmidpREP4-supE was incorporated into the E. coli strain WK6 [(lac-proAB),thi, rpsL, nal^(r); F¹lacI⁴, lacZM15, proA+B+] to demonstrate ability tocontrol expression of surface-exposed proteins. This strain has nochromosomally coded supE gene. The strain was also transformed with theexpression plasmid pASK-INT-EETI-CKSend (see above). The E. coli strain,now transformed with two plasmids, was cultured in parallel trials inthe presence of increasing amounts of IPTG (0, 5, 10, 15, 20, 30, 40,50, 1000 μM). When an absorbance of 0.2 was reached, 0.2 μg/mlanhydracycline [tr. note #8] was added to induce transcription of theIntimin gene. The cells were harvested after one hour of induction. Thecells were labeled successively with anti-Etag antibody, biotinylatedgoat anti-mouse antibody and, finally, with phycoerythrin-coupledstreptavidin (see above). The fluorescence per cell which that causes isproportional to the number of Intimin-fusion proteins exposed on thesurface of the cell. The cellular fluorescence was measured by flowcytometry. As FIG. 8 shows, the intensity of the fluorescence per cell,and thus the number of exposed molecules, is directly correlated withthe amount of IPTG inducer. It can be modulated in other regions.

Example 2 Bacterial Surface Exposure of Protein Fragments, EpitopeMapping, and Isolation of Monospecific Antibodies

[0105] The gene for PMS1 from the yeast S. cerevisiae (Gene BankAccession Number M29688) was used as the model antigen. The gene (2.7kB) was amplified using the PCR primers PMS1up and PMS1lo from S.cerevisiae. The DNA was purified with the Nucleotrap PCR kit (Macheryand Nagel). The DNAse I digestion was carried out in four differenttrial solutions using 1.8*·10⁻², 2,7^(?)·, 10⁻², 3.6*·10⁻² and 0.18 νDNAse I [tr. note #9] in the presence of 2 mM MnCl2. 5 μg DNA was usedper solution. The restriction cutting was stopped by addition of 500 mMEDTA after 10 minutes at room temperature. The resulting fragments wereseparated on a 12.5% polyacrylamide gel after staining with ethidiumbromide. Fragments in the size range of 40-100 bp were cut out. Then thefragments were eluted from the gel by diffusion overnight in TBE buffer.Then any DNAse I that might still be present was removed byphenol/chloroform extraction and ethanol precipitation of the DNA. Afterthat, overhanging ends were filled out with T4-DNA polymerase in thepresence of dATP, dCTP, dGTP and dTTP. Then oligonucleotide hybridlinkers (see FIG. 10) were ligated to them (2.5 molar excess, 16 hoursat 15° C.) and the ligation products were again purified on a 12.5%[poly]acrylamide gel.

[0106] The solution conditions were the same as in the purification ofthe gene fragments. Then the DNA was again extracted withphenol/chloroform and precipitated. The cloning vectorpASK-INT-EETI-CKSend was cut with AvaI/BamHI and purified bycentrifugation in a sucrose gradient (Kolmar, H. & Fritz, H.-J. (1995).Oligonucleotide-directed mutagenesis with single-stranded cloningvectors. In: DNA Cloning 1: A Practical Approach. D. Glover, B. D. Hames(Eds.), IRL Press, Oxford, pages 193-224). A total of 230 ng splitvector was ligated with the fragmented DNA per gene bank, andelectrocompetent BMH 71-18 cells were transformed with it. A collectionof 7,2* 10⁴ independent clones was obtained. The population of bacterialcells was incubated with polyclonal anti-PMS1 rabbit serum afterinduction of the expression of the Intimin fusion gene by addition ofanhydrotetracycline. The specific binding of antibodies toepitope-carrying cells was determined by labeling with biotinylatedanti-rabbit antibodies and incubation with streptavidin-phycoerythrinconjugate. Fluorescence-labeled cells were sorted by FACS and depositedindividually on an agar plate with a MoFlo Cellsorter (Cytomation). DNAwas prepared from a number of clones and the nucleotide sequences offour cloned PMS1 gene fragments were determined. The DNA sequence of theclones involves the bases 259-330 (PMS1_KL3), 1630-1672 (PMS1_KL6),1786-1836 (PMS1_KL7) and 2590-2639 (PMS1_KL10) of the pms1 gene (a totalof 2715 bp). FIG. 11 shows the positions of these epitopes in thepeptide sequence of the PMS1 protein, as well as the translated aminoacid sequence of the cloned PMS1 sequence segment.

[0107] Cultures of the clones PMS_KL6 and PMS_KL7 were grown on the 50ml scale. After one hour of induction by addition of anhydrotetracycline(02 μg/ml) at an absorbance of 0.2, the cells were pelleted, washed withPBS buffer, and resuspended in 500 μl PBS. Then 200 μl MS1 serum wasadded to these bacteria and the suspension was incubated on ice for 45minutes. Then the cells were pelleted and the supernatant was discarded.The cell pellet was resuspended in 200 μl glycine/NaCl (0.2 M glycine,0.145 M NaCl, pH 2.0), resuspended, and incubated for 45 minutes on ice.Following the incubation, the bacteria were centrifuged off. Thesupernatant was made alkaline by addition of 100 μl of 1 M Tris/HCl, pH9. The cells of clone KL6 and KL7 were incubated with the monospecificantibodies obtained in this manner (see FIG. 12).

[0108] This shows that monospecific antibodies can be isolated with thisprocedure.

Example 3 Isolation of Peptides with Affinity to a Specified TargetProtein by Intimin-Based Surface Exposure of Combinatorial PeptideLibraries

[0109] A library of variants of the cystine node protein EETI-II,comprising 28 amino acids, was generated. EETI-II is a trypsin proteaseinhibitor that occurs in the vegetable marrow Ecballium elaterium. Thispeptide is stabilized by three intramolecular disulfide bridges whichproduce a series of surface loops (described in Wentzel et al. (1999),J. Biol. Chem. 274: 21037-21043). A library of EETI-II variants wasgenerated, in which the groups of two loop regions exposed to thesolvent are randomized. The amino acid sequence of the EETI-IIcollection is:

[0110] GCXXXXMRCKQDSDCLAGCVCQVLXPXXSXCG

[0111] (Amino acids in one-letter code. The two loop regions in whichamino acid position(x)s are randomized are indicated in italics.)

[0112] The randomized eeti genes were generated using PCR. Aneeti-ckSend gene (see above) was used as the template. It was amplifiedusing the degenerate primer eti_(—)4+4up and eti_(—)4+4lo. Therandomization of the corresponding codons in the primer sequenceoccurred according to the pattern NNS, in which N represents one of thefour nucleotides and S represents G or C. This selection excluded thestop codons ochre (TAA) and opal (TGA) and reduced the number ofpossible codons from 64 to 32, but they still code for all the aminoacids. The mixture of resulting PCR products was cut with AvaI and BamHIand ligated with the vector fragment pASK-INT-EETI-CKSend cut with theAvaI/BamHI.

[0113] The pASK-INT-EETI-CKSend served as the cloning vector (see FIG.8). This vector was cut with AvaI and BamHI. Then the vector fragmentwas separated from the vector DNA by means of sucrose gradientcentrifugation. The eeti4+4 genes generated by PCR with degeneratedprimers were ligated with the purified vector and the E. coli strainDH5((Hanahn, d. (1983), J. Mol. Biol. 166: 557-580) was transformed withthis solution. Sixteen transformations were done in parallel. All thetransformants were streaked on selective plates containingchloramphenicol (25 μg/ml). After incubation for about 20 hours at 37°C., the colonies were suspended and the cells were stored as aliquots at−80° C. after addition of DMSO (7%). A total of 2* 10⁷ independentclones was generated in this manner.

[0114] 50 ml of liquid culture was inoculated with 10⁹ cells of thestored library to isolate microproteins with affinity toanti-β-lactamase antibodies. On reaching an absorbance of 0.4,anhydrotetracycline was added (0.2 μg/ml) to induce the gene expression.After induction, the cells were fluorescence-labeled. That was done byincubating the cells first for 10 minutes anti-β-lactamase antibodiesand then washing them with PBS to remove unbound protein. Then the cellswere incubated with biotinylated anti-rabbit antibodies and then withstreptavidin-coupled R-phycoerythrin. After labeling the cells wereanalyzed in the Cellsorter, with fluorescent cells sorted out. Thosebacteria which fell between 300 and 1024 in a fluorescence channel weredefined as fluorescent (see FIG. 14).

[0115] Cells sorted out were plated on agar plates containingchloramphenicol (26 μg/ml) and incubated overnight at 37° C. Thecolonies were suspended on the following day and stored as DMSO cultureat −80° C. One aliquot was used to inoculate a fresh 50 ml culture.

[0116] A total of 2* 10⁸ cells was analyzed in the first round. Thosewhich fell into the appropriate fluorescence channel (300-1024), weresorted out. In the second round, cells, which fell into thisfluorescence channel (4.2% in all), were sorted out and then immediatelyresorted twice. These cells (5* 10⁵ events) were plated out and went tothe third round on the next day. Here a significant increase of bacteriafalling into the selected range was recorded (30%). The cells were againsorted and plated so that individual clones could be analyzed on thefollowing day. Fifteen of twenty individual clones showed interactionwith anti-β-lactamase. Plasmid DNA was isolated from 6 of these clones,and the nucleotide sequence of the gene for the EETI-CK variant wasdetermined (FIG. 15).

[0117] Protein sequences from the Intimin family usable within thelimits of this invention are described at the following locations in theliterature:

[0118] 1) Gamma Intimin (Escherichia coli), NCBI GI 3941710, NCBI GI3941712, NCBI GI 3941714, McGraw, E. A., in “Molecular evolution andmosaic structure of alpha, beta Intimins of pathogenic Escherichiacoli”, Mol. Biol. Evol. 16 (1) 12-22 (1999).

[0119] 2) Intimin (attaching and effacing protein, eae protein) NCBI GI1169452, Yu, Ji and Kaper, J. B., in “Cloning and characterization ofthe eae gene of enterohae. Escherichia coli O157:H7”, Mol. Microbiol.6(3), 411-417 (1992).

[0120] 3) eae gene, NCBI GI 384173, Beebakhee, G., et al. In “Cloningand nucleotide sequence of the eae gene homologue enterohemorrhagicEscherichia coli Serotype O157/H7”, FEMS Microbiol. Lett. 91(1), 63-68(1992).

[0121] 4) Intimin (Escherichia coli), NCBI GI 2565325, Voss, E. et al.in “Translocated intimin receptors (Tir) of shiga-toxigenic E. coliisolates belonging to serogroups O26, O111 and O157 with sera frompatients with hemolytic-uremic syndrome and marked sequenceheterogeneity”, Infect. Immun. 66(11), 5580-5586 (1998).

[0122] 5) Intimin (Escherichia coli) NCBI GI 2865299, Elliott, S. J., etal. In “The complete sequence of the locus of enterocyte effacement fromenteropathogenic Escherichia coli E2348/69”, Mol. Microbiol. 28(1), 1-4(1998).

[0123] 6) Beta Intimin (Escherichia coli), NCBI GI 3941718, McGraw etal., loc. cit.

[0124] 7) Intimin (Escherichia coli), NCBI GI 2739264, Deibel, D. et al.In “EspE, a novel secreted protein of attaching and effacing is directlytranslocated into infected host cells, whereas a trypsin-phosphorylated90 kDa protein” [sic] Mol. Microbiol. 28(3), 463-474 (1998).

[0125] 8) Intimin (Escherichia coli) NCBI GI 4388530

[0126] 9) Intimin (Escherichia coli) NCBI GI 4388530

[0127] 10) Intimin type epsilon (Escherichia coli) NCBI GI 6683770

[0128] 11) Intimin NCBI GI 1947048

[0129] 12) Intimin NCBI GI 4106360

[0130] 13) Intimin (Escherichia coli) NCBI GI 6649538

[0131] 14) Intimin (Escherichia coli) NCBI GI 2809548

[0132] 15) Intimin (Escherichia coli) NCBI GI 7384863

[0133] 16) Shares homology with the enteropathogenic E. coli (EPEC)attaching and effacing) gene, putative NCBI GI 304362

[0134] 17) Intimin (Escherichia coli) NCBI GI 7384863.

[0135] Oligonucleotides Used

[0136] [see page 25 in original German text]

1 24 1 32 PRT Artificial Ecballium elaterium, variant; figure 15-1 1 GlyCys Val Met Thr Gly Ile Arg Cys Lys Gln Asp Ser Asp Cys Leu 1 5 10 15Ala Gly Cys Val Cys Gln Val Leu Asn Pro Lys Thr Ser Asn Cys Gly 20 25 302 32 PRT Artificial Ecballium elaterium, variant; figure 15-5 2 Gly CysAsn Arg Ser Leu Ile Arg Cys Lys Gln Asp Ser Asp Cys Leu 1 5 10 15 AlaGly Cys Val Cys Gln Val Leu Asn Pro Pro Thr Ser Asn Cys Gly 20 25 30 332 PRT Artificial Ecballium elaterium, variant; figure 15-8 3 Gly CysTrp Glu Arg Asp Ile Arg Cys Lys Gln Asp Ser Asp Cys Leu 1 5 10 15 AlaGly Cys Val Cys Gln Val Leu His Pro Ser Gln Ser Tyr Cys Gly 20 25 30 432 PRT Artificial Ecballium elaterium, variant; figure 15-9 4 Gly CysVal Thr Ser Leu Ile Arg Cys Lys Gln Asp Ser Asp Cys Leu 1 5 10 15 AlaGly Cys Val Cys Gln Val Leu His Pro Pro Tyr Tyr Asn Cys Gly 20 25 30 532 PRT Artificial Ecballium elaterium, variant; figure 15-3 5 Gly CysVal Ser Ser His Ile Arg Cys Lys Gln Asp Ser Asp Cys Leu 1 5 10 15 AlaGly Cys Val Cys Gln Val Leu His Pro Pro Tyr Gln Asn Cys Gly 20 25 30 632 PRT Artificial Ecballium elaterium, variant; figure 15-7 6 Gly CysMet Asp Thr His Ile Arg Cys Lys Gln Asp Ser Asp Cys Leu 1 5 10 15 AlaGly Cys Val Cys Gln Val Leu Asn Pro Pro Thr Ser Asn Cys Gly 20 25 30 732 PRT Artificial Ecballium elaterium, variant; EETI-II Repertoire 7 GlyCys Xaa Xaa Xaa Xaa Met Arg Cys Lys Gln Asp Ser Asp Cys Leu 1 5 10 15Ala Gly Cys Val Cys Gln Val Leu Xaa Pro Xaa Xaa Ser Xaa Cys Gly 20 25 308 29 DNA Artificial EHEC, variant; oligonucleotide Intilo 1 8 gcgccaattgcgctggcctt ggtttgatc 29 9 48 DNA Artificial EHEC, variant;oligonucleotide Intiminup 9 gcgctctaga taacgagggc aaaaaatgat tactcatggttgttatac 48 10 29 DNA Artificial Escherichia coli, vairant; SupE2_coup10 gcgcgaattc accagaaagc gttgtacgg 29 11 28 DNA Artificial Escherichiacoli, variant; SupE2_Mlu-lo 11 gcgcacgcgt aagacgcggc agcgtcgc 28 12 19DNA Artificial Saccharomyces cerevisiae, variant; PMS1up 12 gcgatgtttcaccacatcg 19 13 19 DNA Artificial Saccharomyces cerevisiae, variant;PMS1lo 13 tcatatttcg taatccttc 19 14 47 DNA ArtificialOligodeoxyribonucleotide to the Amplification and Mutation, artificialDNA-sequence from Ecballium elaterium Trypsin Inhibitor II; eti-4+4up 14gacgcccggg tgcnnsnnsn nsnnsatccg ttgcaaacag gactccg 47 15 60 DNAArtificial Oligodeoxyribonucleotide to the Amplification and Mutation,artificial DNA Sequence from Ecballium elaterium Trypsin Inhibitor II;eti-4+4lo 15 gcgcgcggat ccgcasnnag asnnsnnagg snngagaacc tggcaaacgcagccagccag 60 16 644 DNA Escherichia coli SupE-Eco (1)..(644) 16caccagaaag cgttgtacgg atggggtatc gccaagcggt aaggcaccgg tttttgatac 60cggcattccc tggttcgaat ccaggtaccc cagccatctt cttcgagtaa gcggttcacc 120gcccggttat tggggtatcg ccaagcggta aggcaccggt ttttgatacc ggcattccct 180ggttcgaatc caggtacccc agccatcgaa gaaacaatct ggctacgtag ctcagttggt 240tagagcacat cactcataat gatggggtca caggttcgaa tcccgtcgta gccaccaaat 300tctgaatgta tcgaatatgt tcggcaaatt caaaaccaat ttgttggggt atcgccaagc 360ggtaaggcac cggattctaa ttccggcatt ccgaggttcg aatcctcgta ccccagccaa 420tttattcaag acgcttacct tgtaagtgca cccagttggg gtatcgccaa gcggtaaggc 480accggattct gattccggca ttccgaggtt cgaatcctcg taccccagcc acattaaaaa 540agctcgcttc ggcgagcttt ttgcttttct gcgttcattc aatgtcgaat gcgatgttga 600cacgtcttat ccttcaatgt cggatgcgac gctgccgcgt ctta 644 17 2238 DNAArtificial Hybrid of tetA promotor region ASK75), Intimin EaeA fragmentof EHEC, E-tag epitope, variant from EETI-II (Ecballium elateriumtrypsin Inhibitor) with internal Epitpoe of Sendai Virus 17 tattttaccactccctatca gtgatagaga aaagtgaaat gaatagttcg acaaaaatct 60 agataacgagggcaaaaaat gattactcat ggttgttata cccggacccg gcacaagcat 120 aagctaaaaaaaacattgat tatgcttagt gctggtttag gattgttttt ttatgttaat 180 tagaattcatttgcaaatgg tgaaaattat tttaaattgg gttcggattc aaaactgtta 240 actcatgatagctatcagaa tcgccttttt tatacgttga aaactggtga aactgttgcc 300 gatctttctaaatcgcaaga tattaattta tcgacgattt ggtcgttgaa taagcattta 360 tacagttctgaaagcgaaat gatgaaggcc gcgcctggtc agcagatcat tttgccactc 420 aaaaaacttccctttgaata cagtgcacta ccacttttag gttcggcacc tcttgttgct 480 gcaggtggtgttgctggtca cacgaataaa ctgactaaaa tgtccccgga cgtgaccaaa 540 agcaacatgaccgatgacaa ggcattaaat tatgcggcac aacaggcggc gagtctcggt 600 agccagcttcagtcgcgatc tctgaacggc gattacgcga aagataccgc tcttggtatc 660 gctggtaaccaggcttcgtc acagttgcag gcctggttac aacattatgg aacggcagag 720 gttaatctgcagagtggtga taactttgac ggtagttcac tggacttctt attaccgttc 780 tatgattccgaaaaaatgct ggcatttggt caggtcggag cgcgttacat tgactcccgc 840 tttacggcaaatttaggtgc gggtcagcgt tttttccttc ctgcaaacat gttgggctat 900 aacgtcttcattgatcagga tttttctggt gataataccc gtttaggtat tggtggcgaa 960 tactggcgagactatttcaa aagtagcgtt aacggctatt tccgcatgag gcgctggcat 1020 gagtcataccataagaaaga ctatgatgag cgcccagcaa atggcttcga tatccgtttt 1080 aatggctatctaccgtcata tccggcatta ggcgccaagc tgatatatga gcagtattat 1140 ggtgataatgttgctttgtt taattctgat aagctgcagt cgaatcctgg tgcggcgacc 1200 gttggtgtaaactatactcc gattcctctg gtgacgatgg ggatcgatta ccgtcatggt 1260 acgggtaatgaaaatgatct cctttactca atgcagttcc gttatcagtt tgataaatcg 1320 tggtctcagcaaattgaacc acagtatgtt aacgagttaa gaacattatc aggcagccgt 1380 tacgatctggttcagcgtaa taacaatatt attctggagt acaagaagca ggatattctt 1440 tctctgaatattccgcatga tattaatggt actgaacaca gtacgcagaa gattcagttg 1500 atcgttaagagcaaatacgg tctggatcgt atcgtctggg atgatagtgc attacgcagt 1560 cagggcggtcagattcagca tagcggaagc caaagcgcac aagactacca ggctattttg 1620 cctgcttatgtgcaaggtgg cagcaatatt tataaagtga cggctcgcgc ctatgaccgt 1680 aatggcaatagctctaacaa tgtacagctt actattaccg ttctgtcgaa tggtcaagtt 1740 gtcgaccaggttggggtaac ggactttacg gcggataaga cttcggctaa agcggataac 1800 gccgataccattacttatac cgcgacggtg aaaaagaatg gggtagctca ggctaatgtc 1860 cctgtttcatttaatattgt ttcaggaact gcaactcttg gggcaaatag tgccaaaacg 1920 gatgctaacggtaaggcaac cgtaacgttg aagtcgagta cgccaggaca ggtcgtcgtg 1980 tctgctaaaaccgcggagat gagttcagca cttaatgcca gtgcggttat attttttgat 2040 caaaccaaggccagcgcaat tcctccaacg cccctgggtg cgccggtacc gtatccagat 2100 ccgctggaaccgcgtgccgc ttctggcccc gggtgcgatg gaagcttagg tgatatcgaa 2160 ccatacgattcatcatgcaa acaggactcc gactgcctgg ctggctgcgt ttgcgggccc 2220 aacggtttctgcggatcc 2238 18 23 DNA Artificial Coding strand of linker molecule infigure 10A 18 ccgggtccgg aagcggttcc ggg 23 19 18 DNA Artificial Codingstrand of linker molecule in Figure 10B. 19 taactgactg acccgcag 18 20 23PRT Saccharomyces cerevisiae MISC_FEATURE (1)..(23) epitope in proteinsequence of PMS1, Figure 11 20 Glu Cys Ser Asp Asn Gly Asp Gly Ile AspPro Ser Asn Tyr Glu Phe 1 5 10 15 Leu Ala Leu Lys His Tyr Thr 20 21 13PRT Saccharomyces cerevisiae MISC_FEATURE (1)..(13) epitope in theprotein sequence of PMS1, Figure 11 21 Tyr Phe Asp Ile Asp Gly Glu LysPhe Gln Glu Lys Ala 1 5 10 22 22 PRT Saccharomyces cerevisiaeMISC_FEATURE (1)..(22) epitope in the protein sequence of PMS1; Figure11 22 Asp Ser Ile Tyr Ala Glu Ile Glu Pro Val Glu Ile Asn Val Arg Thr 15 10 15 Pro Leu Lys Asn Ser Arg 20 23 15 PRT Saccharomyces cerevisiaeMISC_FEATURE (1)..(15) epitope in the protein seuqnece PMS1; Figure 1123 Thr Arg Val Val His Asn Leu Ser Glu Leu Asp Lys Pro Trp Asn 1 5 10 1524 108 PRT Escherichia coli MISC_FEATURE (1)..(108) correspondingtranslated amino acid sequence; Figure 2 24 Met Ile Thr His Gly Cys TyrThr Arg Thr Arg His Lys His Lys Leu 1 5 10 15 Lys Lys Thr Leu Ile MetLeu Ser Ala Gly Leu Gly Leu Phe Phe Tyr 20 25 30 Val Asn Asn Ser Phe AlaAsn Gly Phe Asp Gln Thr Lys Ala Ser Ala 35 40 45 Ile Pro Pro Thr Pro LeuGly Ala Pro Val Pro Tyr Pro Asp Pro Leu 50 55 60 Glu Pro Arg Ala Ala SerGly Pro Gly Cys Asp Gly Ser Leu Gly Asp 65 70 75 80 Leu Glu Pro Tyr AspSer Ser Cys Lys Gln Asp Ser Asp Cys Leu Ala 85 90 95 Gly Cys Val Cys GlyPro Asn Gly Phe Cys Gly Ser 100 105

1. Process for exposing peptides and proteins on the surface of hostbacteria, in which one (a) prepares a Gram-negative host bacterium thatis transformed with a vector on which a fused nucleic acid sequence islocalized which is in operative linkage with an exogenously induciblepromoter that (i) codes for a sequence segment which is an Intiminshortened by at least the C3 domain in the carboxyterminal region as theanchoring domain, and (ii) has a nucleic acid sequence segment codingfor the passenger peptide or passenger polypeptide to be exposed, and(b) cultivates the host bacterium under conditions in which thepeptide/polypeptide/protein coded by the nucleic acid segment (ii) isexpressed and exposed on the surface of the host bacterium, whereby thenucleic acid sequence segment (ii) is heterologous with respect to thenucleic acid sequence segment coding for the Intimin membrane anchoringdomain.
 2. Process according to claim 1, characterized in that theshortened Intimin is shortened in the carboxy terminal region of 280amino acids by at least one other of the Intimin domains D0, D1, D2. 3.Process according to claim 1, characterized in that the Intiminanchoring domain used in the external bacterial membrane was derivedfrom the genus [sic/tr. note #10] of the Enterobacteriaceae and is usedin a host bacterium of a genus of the Enterobacteriaceae.
 4. Processaccording to claim 3, characterized in that a fragment of the Intiminfrom enterohemorrhagic E. coli or a variant of it is used as theanchoring domain.
 5. Process according to claim 4, characterized in thata carboxy-terminal shortened variant of the Eae Intimin from hemorrhagicEscherichia coli O157:H7 is used as the anchoring domain.
 6. Processaccording to claim 5, characterized in that the shortened variant of theEae Intimin contains the amino acids 1 to
 659. 7. Process according toclaim 5, characterized in that the shortened variant of the Eae Intimincontains the amino acids 1 to
 753. 8. Process according to claim 1, inwhich the expression of the fusion gene from the nucleic acid sequencesegment coding for the shortened Intimin and the nucleic acid sequencesegment coding for the passenger protein can be controlled, whereby (i)a codon of the nucleic acid sequence coding for the shortened Intiminwhich codes for glutamine is replaced by an amber stop codon (TAG), and(ii) an Escherichia coli host strain is used in which translation of themRNA of the fusion gene is accomplished by providing a controllableamount of suppressor tRNA which allows over-reading of the stop codon inthe translation.
 9. Process according to claim 8, in which theregulation of the expression of the gene for the amber suppressor tRNAtakes place due to the fact that it is placed under control of apromoter which is controllable in its transcription rate.
 10. Processaccording to claim 9, in which the promoter, which is controllable inits transcription rate, is the PLlac promoter.
 11. Process for producinga variant population of surface-exposed peptides/polypeptides/proteinsand for identifying bacteria, which carry thepeptides/polypeptides/proteins with a desired property, in which theprocess has the following steps: (i) production of one or more fusiongenes or fusion gene fragments by cloning the coding sequence of adesired passenger in the continuous reading frame with the codingsequence of the shortened Intimin gene in at least one expressionvector; (ii) variation of the passenger peptide by one of the methodsselected from: intentional site-directed mutagenesis, e. g., through thepolymerase chain reaction (PCR) using oligonucleotides withintentionally exchanged bases, by random mutagenesis usingoligonucleotides with randomly generated base sequences in selectedsequence segments in the PCR, through error-prone PCR, randomlycontrolled chemical or radiation-generated mutagenesis, (iii)introducing at least one vector into the host bacteria which expose thepassenger stably on their surfaces, (iv) expression of the fusion genein the host bacteria, (v) cultivation of the bacteria to produce astable surface-exposed passenger.
 12. Process according to claim 11,which further comprises: (vi) selective enrichment of the bacteria whichcarrier the passenger with the desired properties on the surface. 13.Process according to claims 11 or 12, in which the identification of thebacterium which carries a passenger with a desired binding affinityexposed on the surface is accomplished by binding to an immobilizedand/or labeled binding partner.
 14. Process according to claim 11 or 12,in which a population of bacteria which carry a surface-exposedpassenger with a binding affinity to a binding partner is used as thematrix for affinity chromatography purification of the binding partnersfrom a mixture of substances.
 15. Expression vector for the expressionof a fusion gene under the control of an exogeneously inducible promoterin which there is, in operative linkage with the promoter, a codingsequence for a desired passenger peptide in the continuous reading framedownstream to a coding sequence for an Intimin shortened by at least theD3 domain in the carboxy-terminal region of 280 amino acids s theanchoring domain for the passenger peptide
 16. Expression vectoraccording to claim 15, in which the promoter is selected from the grouplac promoter, ara promoter, tetA promoter.
 17. Gram-negative hostbacterium of the genus [sic/tr. note #10] Enterobactericeae, transformedwith at least one expression vector according to claim 15.